Separation of biological substance may be classified as the most fundamental operation in biology, genetics and medical science etc. for not only analysis of substance but also cultivation of cells, and identification and amplification of DNA. Frontier medical research is competitively conducted in regenerative medicines using stem cells. The stem cells include embryonic stem cells obtained from blastocyst of initial stage of generation and adult stem cells obtained from adult or placenta whose generation process has been terminated.
The adult stem cells are obtained by three principal methods of: firstly using bone marrow; secondly, umbilical cord blood; and thirdly, peripheral blood (mesenchymal blood) which enables easy harvesting of the adult stem cells. In all these methods, blood is first separated into major components of erythrocyte, leukocyte, blood platelet and blood plasma by means of centrifugation, followed by an operation of separating the stem cell (mononuclear).
In this connection, conventional centrifugation methods may be summarized as follows. When a contained solution of various mixed substances, particularly, collected blood of human being or animal is centrifuged, erythrocyte, leukocyte, blood platelet and other blood plasma in the blood are orderly positioned in layers in the order of weight with the heaviest fraction layered at the bottom of the container due to their inherent difference in specific gravity.
In the initial process of transferring the peripheral blood extracted through syringe needle to the centrifuge container, a stopper of the centrifuge container is opened, the needle of the syringe is positioned inside the centrifuge container, the peripheral blood in the syringe is passed to the centrifuge container, and thereafter the needle is removed from the centrifuge container and the opened centrifuge container is closed with the stopper. The centrifuge container is subsequently positioned in bucket of the centrifugal separator, and the separator is operated by setting desired time, rotational speed (rpm) or acceleration of gravity (g).
The erythrocyte typically has the largest specific gravity among the components of the blood and thus is collected in the bottom portion of the centrifuge container, and the erythrocyte account for 47% of the whole blood for man and 42% for woman. Positioned above the erythrocyte are the leukocyte and blood platelet which account for about 1% of the whole blood, which further includes monocytes or stem cells of the peripheral blood.
The remainder thereabove is serum and fibrinogen referred to as blood plasma. The centrifuge container that completed the centrifugation is recovered and the stopper is then reopened so that a desired substance may be extracted from among the substances separated due to the difference in specific gravity of the component substances, by means of a pipette or capillary tube or needle, and thereafter the remaining components are separately stored or discarded or re-cycled.
In recent times, there has been strong public interest in autologous blood cell regeneration treatment (hereinafter called PRP treatment) which is the collection of PRP from a single patient by way of the aforementioned separation and injection back to the same patient at the lesion where tissue has been damaged to regenerate the tissue, whereby healing the wound. The PRP treatment is widely applied to the field of treatment such as treatment for regenerating the damaged cartilage and treatment for regenerating damaged skin tissue such as acne marks so as to improve the skin condition.
The platelets in the blood and are known to be rich with growth factors, and are also known to cause cell proliferation in the human body, produce collagen, leads to regeneration of angiogenic cells and serves to heal wounds.
One microliter of the human blood contains about 100,000-200,000 or so platelets, and in order for a PRP treatment to be effective, they are concentrated to have 1 million or more counts of platelets per microliter. Such concentration of platelets may be performed through stratifying collected blood by using a centrifugal separator to have typical blood layered with the red blood cells or erythrocytes forming the bottommost layer, a dark yellow buffy coat layer thereon formed of leukocytes and platelets, and pale yellow plasma forming the uppermost layer.
The respective blood layers separated as above are, on average, erythrocyte layer occupying about 45% in volume ratio, the buffy coat layer by 1%, and the plasma layer by 54%. The PRP treatment utilizes the platelet-rich buffy coat layer extracted from the separated blood as described above wherein the mere 1% volume ratio makes it difficult for a conventional blood collection kit to accurately extract the buffy coat alone.
Korean Patent Application Publication No. 10-2010-0116106, entitled “Assembly, device kit and method for preparing platelet-rich plasma” (applicant: Estar Technologies Ltd.) discloses a centrifuge test tube for concentrating platelets of whole blood extracted from a patient to obtain fractions thereof. However, the centrifuge tube in the form of a cylinder with a uniform inner diameter is not apt for accurately extracting the buffy coat fraction only.
Korean Patent Application Publication No. 10-2011-0045980, entitled “Kit of centrifuge separation and methods for centrifuging using the same” (applicant: GLOTECH) discloses collecting whole blood or bodily fluids and then providing a centrifuge separation kit installed with a syringe to undergo centrifugation for immediately collecting target substances such as platelet-rich plasma or stem cells. The centrifuge separation kit still maintains a centrifuge separation tube in the form of a cylinder with a uniform inner diameter, which is susceptible to undesirable accompaniment of separation gel or red blood cells to the target buffy coat during the collection process.
Therefore, Korean Patent Application Publication No. 10-2012-0089723 filed by the present applicants discloses a centrifuge tube provided with a narrowed chamber for separately forming buffy coat therein and at least one rib at an upper chamber and at an intermediate chamber to prevent lateral fluid movements from occurring especially in fixed angle centrifuges and thereby separates substances in the tube accurately. However, the centrifuge tube lacks a means for controlling the buffy coat layer once it is received in a lower chamber of the tube.
Furthermore, the conventional separator kits have caps that involve manual uncapping, injection of blood from a syringe and then recapping with a human hand, which is seriously insanitary and leaves the blood susceptible to contamination through airborne infection.